The present invention relates to an immunoassay technique of magnetically detecting antigen-antibody reactions by using magnetic particles and a superconducting quantum interference device (SQUID) magnetic sensor.
Today, requirements are increasing for high-sensitivity detection of such objects as various pathogenic bacteria, cancer cells, DNAs and environmentally harmful substances by immune reactions, and many attempts have been made to develop immunoassay systems to meet these requirements. Common immunoassay methods include an optical technique by which a detective antibody which selectively combines with the antigen to be detected is marked with an optical marker such as a fluorescent enzyme or the like, and optical signals from the optical marker indicating any antigen-antibody combining reaction are detected to identify the type and quantity of the antigen involved. However, optical methods are not adequate in the sensitivity of detection, and require a step to was away uncombined optical markers.
With a view to achieving a higher level of detection sensitivity than optical methods, magnetic methods by which antigen-antibody reactions are detected by using magnetic particles and a SQUID magnetic sensor have come to be proposed in recent years. By such a magnetic method, an antibody for detection use magnetically marked with magnetic particles (hereinafter referred to as magnetic marker) is detected with an extremely sensitive SQUID magnetic sensor.
Methods of detecting magnetic markers will be described below. Methods based on (1) measurement of magnetic susceptibility, (2) measurement of magnetic relaxation or (3) measurement of remanent magnetism are proposed. The following paragraphs will describe (1) through (3).
(1) Method Using Measurement of Magnetic Susceptibility
A DC magnetic field to magnetize magnetic markers is applied in a direction at a right angle to the direction of magnetic flux detection of a SQUID magnetic sensor, and variations in magnetic field generated by the magnetic markers moving within the area of magnetic flux detection of the SQUID magnetic sensor are measured (see, for instance, Japanese Patent Application Laid-Open No. 2001-33455, Keiji Enpuku, the Journal of the (Japanese) Society of Applied Physics, Vol. 73, No. 1, 28 (2004) (in Japanese); K. Enpuku, et al., Jpn. J. Appl. Phys. 38, L1102 (1999); and K. Enpuku, et al., IEEE Trans. Appl. Supercond. 11, 661 (2001)).
Alternatively, an AC magnetic field is applied to a magnetic marker, and the resultant signal is picked up with a SQUID magnetic sensor to detect an antigen-antibody reaction (see, for instance, Japanese Patent Application Laid-Open No. 2001-133458).
(2) Method of Measuring Magnetic Relaxation
The magnetic relaxation from immediately after applying a pulse magnetic field of 1 mT to magnetic markers until one second afterwards is measured. The measurement is carried out in a solution in which uncombined magnetic markers coexist, and combined magnetic markers are detected (see, for instance, Keiji Enpuku, the Journal of the (Japanese) Society of Applied Physics, Vol. 73, No. 1, 28 (2004) (in Japanese); Y. R. Chemla, et al., Proc. National Acad. Sciences of U.S.A. 97, 14268 (2000); A. Haller, et al., IEEE Trans. Appl. Supercond. 11, 1371 (2001); and, S. K. Lee, et al., Appl. Phys. Lett. 81, 3094 (2002)).
A method of quantitatively detecting samples in liquid and solid phases by measuring magnetic relaxation, and chemical compounds for detection by measuring magnetic relaxation, their analysis and their use in immuno-magnetography are also reported (see, for instance, International Application Publication No. 10-513551).
(3) Method of Measuring Remanent Magnetism
When magnetic particles grow in size, the remanent magnetism of the magnetic particles is no longer relaxed. A magnetic field of around 0.1 T is applied to a magnetic marker in a position away from a SQUID magnetic sensor to cause remanent magnetization to occur in the magnetic marker. After that, a substrate mounted with a sample is shifted to have its remanent magnetism measured by the SQUID magnetic sensor (see, for instance, Keiji Enpuku, the Journal of the (Japanese) Society of Applied Physics, Vol. 73, No. 1, 28 (2004); R. Kotitz, et al., IEEE Trans. Appl. Supercond. 7, 3678 (1997); and K. Enpuku, et al., IEEE Trans. Appl. Supercond. 13, 371 (2003)).
A method for quantitative detection of analyzed objects in liquid and solid phases, compounds suitable for this purpose and its use in analytical chemistry is reported (see, for instance, International Application Publication No. 11-508031).
Another report concerns the possibility of high-sensitivity detection of magnetic signals by a SQUID magnetic sensor by cooling magnetic markers, after their magnetization, to restrain the Brownian motion of magnetic particles (see, for instance, Japanese Patent Application Laid-Open No. 2003-207511).
According to still another report, a configuration having a rotating body on which a sample is arranged, a magnet for magnetizing magnetic markers when the sample on the rotating body is rotated and a pickup coil made of a normal conducting member is used for magnetizing the magnetic markers in every rotation and detecting the magnetic field in the position of the pickup coil (see, for instance, Japanese Patent Application Laid-Open No. 2003-35758).
Incidentally, yet other reports concern devices for measuring magnetic particles by using an induction coil as the magnetic sensor instead of a SQUID sensor (see, for instance, International Application Publication Nos. 2001-524675 and 2003-515743).
Specific examples of magnetic method to detect antigen-antibody reactions will be described below (see, for instance, Keiji Enpuku, the Journal of the (Japanese) Society of Applied Physics, Vol. 73, No. 1, 28 (2004).
FIG. 1 schematically illustrate an example of procedure of conventional magnetic immunoassay method using antigen-antibody reactions.
As shown in FIG. 1A, fixed antibodies 101 are fixed to the bottom 102 of a sample container. Then, when a living sample is injected into the sample container, antigens 103 contained in the living sample are selectively combined with the fixed antibodies 101 by antigen-antibody reaction as shown in FIG. 1B. Next, when magnetic markers which mark the antigens 103 are injected into the sample container, they are combined by antigen-antibody reaction with the antigens 103 combined with the fixed antibodies 101 as shown in FIG. 1C. Uncombined magnetic markers 104b which are not combined with the antigens 103 are moved at random by Brownian motion in the solution.
The magnetic markers combined with the antigens 103 by antigen-antibody reaction are denoted by reference numeral 104a. When a magnetic field is applied to the sample in this state in the direction indicated by an arrow in FIG. 1D, the magnetic markers 104a combined with the antigens 103 are magnetized and, even after the applied magnetic field disappears, magnetic signals proportional to the quantity of the antigens 103 are generated by the remanent magnetism of the magnetic markers 104a. By detecting these magnetic signals with the pickup coil 105 of a high-sensitivity SQUID magnetic sensor, the quantity of the antigens to be assessed can be measured.
The uncombined magnetic markers 104b are also magnetized, these magnetic signals from the uncombined magnetic markers 104b are cancelled as these markers are moving at random in the solution, resulting a sum of 0. Thus it is not necessary to wash off the uncombined magnetic markers. Considering that immunoassay using optical markers indispensably needs a washing step, the absence of this washing step is one of the remarkable advantages of immunoassay using magnetic markers.
FIG. 2 schematically illustrates an example of magnetic marker used in a magnetic immunoassay method according to the prior art. The magnetic marker has a structure in which a magnetic particle 201 enveloped in a macromolecule 202 is combined with an antibody 203 for detection use. As the magnetic particle 201, an Fe3O4 particle of 25 nm in diameter is used. After the macro-monomer is adsorbed by the magnetic particle 201, the structure in which the magnetic particle 201 is enveloped in the macromolecule 202 is realized by subjecting the monomer and the cross-linker to radical copolymerization in a tetrahydrofuran solvent. The diameter of the particle combining the macromolecule 202 and the magnetic particle 201 is about 80 nm.
FIG. 3 illustrates an example of result of immunoassay using remanent magnetism according to the prior art, wherein the relationship between the weight w (pg) of an antigen (IL8: interleukin 8) and a signal magnetic flux Φs is shown. The weight of the antigen was varied from 0.1 pg to 150 pg, and a good correlation was observed in this range between the two factors. In this experiment, as shown in FIG. 3, the antigen (IL8) was detected down to 0.1 pg. As the molecular weight of IL8 is 10,000, 0.1 pg corresponds to 10 atto mol. Since the quantity of antigen and the signal magnetic flux from the magnetic marker combined with the antigen manifests a certain correlation, the antigen can be detected with high sensitivity by detecting the signal magnetic flux from the magnetic marker with a SQUID, which is a high-sensitivity magnetic sensor.